Lipophorin receptor-mediated lipoprotein endocytosis in insect fat body cells
2003; Elsevier BV; Volume: 44; Issue: 8 Linguagem: Inglês
10.1194/jlr.m300022-jlr200
ISSN1539-7262
AutoresDennis Van Hoof, Kees W. Rodenburg, Dick J. Van der Horst,
Tópico(s)Antioxidant Activity and Oxidative Stress
ResumoHigh-density lipophorin (HDLp) in the circulation of insects is able to selectively deliver lipids to target tissues in a nonendocytic manner. In Locusta migratoria, a member of the LDL receptor family has been identified and shown to mediate endocytosis of HDLp in mammalian cells transfected with the cDNA of this receptor. This insect lipophorin receptor (iLR) is temporally expressed in fat body tissue of young adult as well as larval locusts, as shown by Western blot analysis. Fluorescence microscopy revealed that fat body cells internalize fluorescently labeled HDLp and human receptor-associated protein only when iLR is expressed. Expression of iLR is down-regulated on Day 4 after an ecdysis. Consequently, HDLp is no longer internalized. By starving adult locusts immediately after ecdysis, we were able to prolong iLR expression. In addition, expression of the receptor was induced by starving adults after down-regulation of iLR.These results suggest that iLR mediates endocytosis of HDLp in fat body cells, and that expression of iLR is regulated by the demand of fat body tissue for lipids. High-density lipophorin (HDLp) in the circulation of insects is able to selectively deliver lipids to target tissues in a nonendocytic manner. In Locusta migratoria, a member of the LDL receptor family has been identified and shown to mediate endocytosis of HDLp in mammalian cells transfected with the cDNA of this receptor. This insect lipophorin receptor (iLR) is temporally expressed in fat body tissue of young adult as well as larval locusts, as shown by Western blot analysis. Fluorescence microscopy revealed that fat body cells internalize fluorescently labeled HDLp and human receptor-associated protein only when iLR is expressed. Expression of iLR is down-regulated on Day 4 after an ecdysis. Consequently, HDLp is no longer internalized. By starving adult locusts immediately after ecdysis, we were able to prolong iLR expression. In addition, expression of the receptor was induced by starving adults after down-regulation of iLR. These results suggest that iLR mediates endocytosis of HDLp in fat body cells, and that expression of iLR is regulated by the demand of fat body tissue for lipids. Whereas mammals rely on a wide array of lipoproteins with different compositions and functions (1Frayn K.N. Lipoprotein metabolism.in: Snell K. Metabolic Regulation: A Human Perspective. Portland Press, London1996: 197-217Google Scholar), insects make use of a single type of lipoprotein, high-density lipophorin (HDLp) (2Van der Horst D.J. Van Hoof D. Van Marrewijk W.J.A. Rodenburg K.W. Alternative lipid mobilization: the insect shuttle system.Mol. Cell. Biochem. 2002; 239: 113-119Crossref PubMed Scopus (91) Google Scholar), to effect the transport of lipids through the circulation. HDLp comprises diacylglycerol and phospholipids as major lipid classes. The protein matrix consists of two nonexchangeable apolipoproteins, apolipophorin I (apoLp-I) and apoLp-II, which are derived from a common precursor protein through posttranslational cleavage (3Weers P.M.M. Van Marrewijk W.J.A. Beenakkers A.M.T. Van der Horst D.J. Biosynthesis of locust lipophorin. Apolipophorins I and II originate from a common precursor.J. Biol. Chem. 1993; 268: 4300-4303Abstract Full Text PDF PubMed Google Scholar, 4Bogerd J. Babin P.J. Kooiman F.P. Andre M. Ballagny C. Van Marrewijk W.J.A. Van der Horst D.J. Molecular characterization and gene expression in the eye of the apolipophorin II/I precursor from Locusta migratoria.J. Comp. Neurol. 2000; 427: 546-558Crossref PubMed Scopus (27) Google Scholar). Sequence and domain structure analyses indicate that this precursor protein is homologous to mammalian apolipoprotein B-100 (apoB-100), the nonexchangeable protein component of VLDL and its resulting LDL, and that both proteins have emerged from an ancestral gene (5Babin P.J. Bogerd J. Kooiman F.P. Van Marrewijk W.J.A. Van der Horst D.J. Apolipophorin II/I, apolipoprotein B, vitellogenin, and microsomal triglyceride transfer protein genes are derived from a common ancestor.J. Mol. Evol. 1999; 49: 150-160Crossref PubMed Scopus (178) Google Scholar, 6Mann C.J. Anderson T.A. Read J. Chester S.A. Harrison G.B. Kochl S. Ritchie P.J. Bradbury P. Hussain F.S. Amey J. Vanloo B. Rosseneu M. Infante R. Hancock J.M. Levitt D.G. Banaszak L.J. Scott J. Shoulders C.C. The structure of vitellogenin provides a molecular model for the assembly and secretion of atherogenic lipoproteins.J. Mol. Biol. 1999; 285: 391-408Crossref PubMed Scopus (170) Google Scholar, 7Segrest J.P. Jones M.K. De Loof H. Dashti N. Structure of apolipoprotein B-100 in low density lipoproteins.J. Lipid Res. 2001; 42: 1346-1367Abstract Full Text Full Text PDF PubMed Google Scholar). HDLp is secreted by the insect fat body, an organ combining many of the functions of mammalian liver and adipose tissue (8Locke M. Microscopic anatomy of invertebrates.in: Harrison F.W. Locke M. Insecta. Vol. 11B. Wiley-Liss, New York1998: 641-686Google Scholar). Similar to mammalian adipose tissue, the fat body retains large intracellular lipid depots that provide the fuel for energy-demanding tissues. Circulatory HDLp is able to take up lipids released from the fat body cells and to selectively unload its lipid cargo at target tissues without endocytosis and lysosomal degradation, and thus functions as a reuseable shuttle [as reviewed in refs. (2Van der Horst D.J. Van Hoof D. Van Marrewijk W.J.A. Rodenburg K.W. Alternative lipid mobilization: the insect shuttle system.Mol. Cell. Biochem. 2002; 239: 113-119Crossref PubMed Scopus (91) Google Scholar, 9Van der Horst D.J. Weers P.M.M. Van Marrewijk W.J.A. Lipoproteins and lipid transport.in: Stanley-Samuelson D.W. Nelson D.R. Insect Lipids: Chemistry, Biochemistry and Biology. University of Nebraska Press, Lincoln and London1993: 1-24Google Scholar, 10Soulages J.L. Wells M.A. Lipophorin: The structure of an insect lipoprotein and its role in lipid transport in insects.Adv. Protein Chem. 1994; 45: 371-415Crossref PubMed Google Scholar, 11Ryan R.O. Van der Horst D.J. Lipid transport biochemistry and its role in energy production.Annu. Rev. Entomol. 2000; 45: 233-260Crossref PubMed Scopus (159) Google Scholar)]. In spite of the concept of selective lipid transfer mediated by HDLp, a novel member of the LDL receptor (LDLR) family was identified in Locusta migratoria (12Dantuma N.P. Potters M. De Winther M.P. Tensen C.P. Kooiman F.P. Bogerd J. Van der Horst D.J. An insect homolog of the vertebrate very low density lipoprotein receptor mediates endocytosis of lipophorins.J. Lipid Res. 1999; 40: 973-978Abstract Full Text Full Text PDF PubMed Google Scholar). The domain structure composition of this insect's lipophorin receptor (iLR) is identical to that of the mammalian VLDL receptor (VLDLR), and both have eight consecutive ligand binding repeats. A similar receptor was found in the mosquito Aedes aegypti (13Cheon H.M. Seo S.J. Sun J. Sappington T.W. Raikhel A.S. Molecular characterization of the VLDL receptor homolog mediating binding of lipophorin in oocyte of the mosquito Aedes aegypti.Insect Biochem. Mol. Biol. 2001; 31: 753-760Crossref PubMed Scopus (82) Google Scholar). In a stably transfected Chinese hamster ovary (CHO) cell line, locust iLR was shown to bind and internalize HDLp specifically, but not human LDL (14Van Hoof D. Rodenburg K.W. Van der Horst D.J. Insect lipoprotein follows a transferrin-like recycling pathway that is mediated by the insect LDL receptor homologue.J. Cell Sci. 2002; 115: 4001-4012Crossref PubMed Scopus (42) Google Scholar). In contrast to the lysosomal fate of ligands internalized by mammalian lipoprotein receptors, endocytosed HDLp was observed to escape from degradation after iLR-mediated endocytosis. Both the occurrence of iLR in the insect (12Dantuma N.P. Potters M. De Winther M.P. Tensen C.P. Kooiman F.P. Bogerd J. Van der Horst D.J. An insect homolog of the vertebrate very low density lipoprotein receptor mediates endocytosis of lipophorins.J. Lipid Res. 1999; 40: 973-978Abstract Full Text Full Text PDF PubMed Google Scholar, 13Cheon H.M. Seo S.J. Sun J. Sappington T.W. Raikhel A.S. Molecular characterization of the VLDL receptor homolog mediating binding of lipophorin in oocyte of the mosquito Aedes aegypti.Insect Biochem. Mol. Biol. 2001; 31: 753-760Crossref PubMed Scopus (82) Google Scholar) and its functioning in a mammalian cell line (14Van Hoof D. Rodenburg K.W. Van der Horst D.J. Insect lipoprotein follows a transferrin-like recycling pathway that is mediated by the insect LDL receptor homologue.J. Cell Sci. 2002; 115: 4001-4012Crossref PubMed Scopus (42) Google Scholar) suggested that internalization of HDLp via receptor-mediated endocytosis may be a physiologically relevant process (15Dantuma N.P. Pijnenburg M.A. Diederen J.H.B. Van der Horst D.J. Developmental down-regulation of receptor-mediated endocytosis of an insect lipoprotein.J. Lipid Res. 1997; 38: 254-265Abstract Full Text PDF PubMed Google Scholar). Using fluorescence microscopy, in this study we demonstrate that fat body tissue of young adult and larval locusts is able to internalize fluorescently labeled HDLp via iLR. In addition, similar to mammalian VLDLR, this receptor appears capable of internalizing human receptor-associated protein (RAP). On Day 4 after the energy-consuming process of ecdysis, expression of iLR drops below detectable levels in young adult and larval locusts. Fat body tissue excised from these insects has lost the ability to endocytose HDLp. Down-regulation of iLR was postponed when adults were starved immediately after ecdysis. In addition, starving adult locusts after down-regulation of iLR induced expression of the receptor. Taken together, these results suggest that iLR mediates endocytosis of HDLp in insect cells, and provide evidence for regulation of iLR expression under specific physiological conditions. The endocytic property of iLR is compared with that of VLDLR. In addition, the proposed lipoprotein recycling function observed in mammalian cells as well as its possible role in lipid storage in insects are discussed. Precision protein standards prestained broad range marker (Bio-Rad); alkaline phosphatase-conjugated affinipure goat-anti-rabbit IgG, DiI(C18(3)) (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine percholate), and Oregon Green 488 (OG) carboxylic acid (Molecular Probes); 4′,6′-diamidino-2′-phenylindole (DAPI) (Roche Diagnostics); leupeptin, aprotinin, and BSA (Sigma); 125I[iodine] (3.9 GBq/ml; Amersham Pharmacia Biochem); Oil Red O (Chroma); Coomassie brilliant blue (Serva); trypsin-EDTA (Invitrogen); and chloramine-T (Merck) were obtained from commercial sources. HDLp was isolated from locust hemolymph by ultracentrifugation (14Van Hoof D. Rodenburg K.W. Van der Horst D.J. Insect lipoprotein follows a transferrin-like recycling pathway that is mediated by the insect LDL receptor homologue.J. Cell Sci. 2002; 115: 4001-4012Crossref PubMed Scopus (42) Google Scholar). Membrane proteins of wild-type CHO and iLR-transfected CHO [CHO(iLR)] cells were isolated as described by Van Hoof, Rodenburg, and Van der Horst (14Van Hoof D. Rodenburg K.W. Van der Horst D.J. Insect lipoprotein follows a transferrin-like recycling pathway that is mediated by the insect LDL receptor homologue.J. Cell Sci. 2002; 115: 4001-4012Crossref PubMed Scopus (42) Google Scholar). Polyclonal rabbit-anti-iLR 9218 antibody was raised against a synthetic peptide representing the unique C-terminal 19 amino acids (865–883) of iLR (14Van Hoof D. Rodenburg K.W. Van der Horst D.J. Insect lipoprotein follows a transferrin-like recycling pathway that is mediated by the insect LDL receptor homologue.J. Cell Sci. 2002; 115: 4001-4012Crossref PubMed Scopus (42) Google Scholar), and polyclonal rabbit-anti-iLR 2189/90 antibody was raised against a synthetic peptide representing the unique N-terminal 20 amino acids (34–53) of the first cysteine-rich repeat of iLR. Human RAP was a generous gift from Dr. Michael Etzerodt (IMSB, Aarhus University, Aarhus, Denmark). Insects were reared under crowded conditions in a temperature-controlled environment at 30°C with a relative humidity of 40% and a 12 h light-dark cycle. Immediately after ecdysis, male and female fifth instar (L5) larvae were transferred to separate cages to obtain synchronized larval fat body. The same procedure was used to obtain synchronized adult male and female locust fat body after the imaginal ecdysis. When starved, individual animals were transferred to separate cages and given access to water to prevent dehydration. HDLp (1 mg/ml) was fluorescently labeled in PBS with 50 μl/ml DiI in DMSO (3 μg/μl) at 37°C under continuous stirring for 2.5 h. HDLp and RAP (1 mg/ml) were labeled with 20 μl/ml OG dissolved in DMSO (1 μg/μl) at room temperature under continuous stirring for 1 h according to the manufacturer's instructions. DiI and OG-labeled HDLp (DiI-HDLp and OG-HDLp, respectively) were purified with Sephadex G-25 PD-10 columns (Amersham Pharmacia Biotech) to separate fluorescently labeled ligand from free fluorescent label and replace the PBS by incubation medium (10 mM HEPES, 50 mM NaCl, 10 mM KCl, 5 mM CaCl2, and 2 mM MgSO4; pH 7.4). OG-labeled RAP (OG-RAP) was dialyzed against incubation medium using standard cellulose membrane (Medicell International). Fat body tissue was incubated with 10 μg/ml DiI-HDLp, 25 μg/ml OG-HDLp, or 3.6 μg/ml OG-RAP for 30 min at 32°C for endocytic uptake. Tissue was rinsed in incubation medium and immediately fixed in 4% paraformaldehyde diluted in PBS for 30 min at room temperature. Where indicated, prior to fixation, fat body tissue was incubated with 0.05% trypsin in 0.35 mM EDTA for 5 min at room temperature, and washed thoroughly in incubation medium. For cell surface binding, fat body tissue was incubated with fluorescently labeled HDLp for 1 h at 4°C, thoroughly washed in incubation medium, and fixed as described. For endocytosis of surface-bound HDLp, fat body tissue was preincubated with OG-HDLp for 30 min at 4°C, thoroughly washed, and then incubated in medium without fluorescently labeled HDLp for 30 min at 32°C, followed by fixation as described. After fixation, fat body tissue was incubated with 0.25 μg DAPI per ml PBS for 30 min at room temperature to stain the nuclei of the cells. Coverslips with fixed tissue were mounted in Mowiol and examined on a light and fluorescence Axioscop microscope (Zeiss) with a Hg HBO-50 lamp and a Plan-Neofluar 100×/1.30 oil lens. Using UV and fluorescein isothiocyanate-tetramethylrhodamine isothiocyanate filters, digital images were recorded with a DXM 1200 digital camera and ACT-1 version 2.00 software (Nikon). Images of centrally localized nuclei and peripherally distributed endocytic vesicles of the same area were obtained sequentially at their respective confocal planes. Corresponding images of nuclei and vesicles were subsequently processed and merged using PaintShop Pro 7.00 (Jasc Software). Fat body tissue from male and female larvae and adult locusts was excised in incubation medium containing protease inhibitors. Fat body tissue of three to five individuals was pooled and fractionated by thoroughly resuspending and vortexing, and kept on ice during the following purification steps. Samples were centrifuged for 10 min at 10°C at 15,000 g to separate the fractionated cells from the released lipids. The fat cake was removed with a toothpick and the supernatant was discarded, after which the pellet was resuspended in protease inhibitor-containing incubation medium. The samples were centrifuged again at 15,000 g for 10 min at 10°C, after which the supernatant was removed and the remaining lipids were discarded with a tissue. The lipid-depleted pellets were resuspended in 40 μl to 80 μl 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) buffer (20 mM HEPES, 124 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 2.5 mM Na2HPO4, 1.2 mM MgSO4, 1 mM EDTA, 0.1 mM benzamidine, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and 1% CHAPS) to resuspend the pellet. The suspension was incubated for 10 min on ice, and spun down at 15,000 g for 10 min at 10°C. Supernatant containing 5.0 μg of total membrane protein was transferred to a clean Eppendorf tube and either heated for 5 min at 95°C in Laemmli buffer (16Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (207208) Google Scholar) or directly dissolved in modified Laemmli buffer (containing 0.025% SDS and no disulfide bond-reducing reagents) prior to separation by SDS-PAGE in a 10% polyacrylamide gel. The separated membrane proteins were transferred to polyvinylidene fluoride (PVDF) membrane (Millipore) and incubated with rabbit anti-iLR 9218 (1:2,000, v/v) or 2189/90 (1:100, v/v) antibody for 2 h, followed by 1 h alkaline phosphatase-coupled goat anti-rabbit incubation. Bound second antibody was visualized by incubating the blot in TSM buffer containing 100 mM Tris-HCl, 100 mM NaCl, 10 mM MgAc2, 50 μg/ml p-nitro blue tetrazolium chloride (Boehringer Mannheim), and 25 μg/ml 5-bromo-4-chloro-3-indoyl-phosphate p-toluidine (BCIP; Roche Diagnostics) (pH 9.0). 125I-RAP was prepared using chloramine-T according to Rodenburg et al. (17Rodenburg K.W. Kj⊘ller L. Petersen H.H. Andreasen P.A. Binding of urokinase-type plasminogen activator/plasminogen activator inhibitor-1 complex to endocytosis receptors α2-macroglobulin receptor/low density lipoprotein receptor-related protein and very low density lipoprotein receptor involves basic residues in the inhibitor.Biochem. J. 1998; 329: 55-63Crossref PubMed Scopus (87) Google Scholar), resulting in a specific labeling activity of ∼45,000 cpm/ng protein. PVDF membrane, containing 20.0 μg per lane of total membrane proteins that were separated by SDS-PAGE under nonreducing conditions, was incubated overnight with 12 nM 125I-RAP in binding buffer [20 mM HEPES (pH 7.4), 50 mM NaCl, 2.5 mM CaCl2, and 0.5% (w/v) BSA], after which the blot was washed several times with binding buffer. RAP binding was detected using a PhosphorImager (Molecular Dynamics), and visualized using MD ImageQuant software version 3.3 (Molecular Dynamics). RAP receptor binding was quantified by determining the radioactivity in those parts of the ligand blots that corresponded to receptor-bound complexes using the data from the PhosphorImager. In contrast to the mechanism by which HDLp selectively unloads lipids at target tissues, endocytosis of HDLp may provide an alternative mechanism for the uptake of lipid components in fat body cells. Therefore, HDLp was fluorescently labeled with DiI to visualize the lipoprotein after incubation of fat body tissue that was excised from young adult male locusts within 24 h of ecdysis. Incubation of fat body tissue with incubation medium containing DiI-HDLp resulted in a punctate staining pattern characteristic for endocytosis (Fig. 1A, red dots). To investigate whether, in addition to the lipid, the apolipoprotein component was also internalized, the amine-reactive probe OG was used to label apoLp-I and -II. Analogous incubation conditions with OG-HDLp resulted in a similar endocytic uptake pattern (Fig. 1B, green dots). Scanning vertically through incubated fat body tissue revealed that HDLp-containing vesicles are peripherally localized in the cells (Fig. 1C). Treatment of tissue with trypsin prior to fixation did not alter the punctate staining pattern, verifying that HDLp is encapsulated in membranes (i.e., endocytic vesicles; Fig. 1D). The trophocyte, or adipocyte, is the main cell type that constitutes the fat body, and is used for storage of lipids and glycogen (8Locke M. Microscopic anatomy of invertebrates.in: Harrison F.W. Locke M. Insecta. Vol. 11B. Wiley-Liss, New York1998: 641-686Google Scholar). The size and shape of trophocytes are predominantly determined by the lipid droplets that fill up almost the entire intracellular space (Fig. 1E, F). As a result, cytoplasm is mainly situated peripherally below the cell surface and between the lipid droplets, whereas the nuclei are predominantly localized in the cell center (Fig. 1G). Incubation of young adult female fat body tissue with DiI-HDLp (data not shown) or OG-HDLp (Fig. 1H) resulted in staining patterns identical to those observed with male fat body tissue, which suggests an uptake mechanism that is present in both sexes. In contrast, fat body tissue excised from adults on Day 4 or later after ecdysis remained devoid of fluorescently stained endocytic vesicles when incubated with DiI-HDLp or OG-HDLp (Fig. 1I and J, respectively). These findings suggest a down-regulation of the ability to internalize HDLp via endocytosis that occurs on Day 4 after imaginal ecdysis. To investigate the involvement of a receptor in HDLp endocytosis, fat body tissue was incubated at 4°C, at which receptor-mediated endocytosis is inhibited, whereas cell surface binding still occurs (18Dunn W.A. Hubbard A.L. Aronson N.N. Low temperature selectively inhibits fusion between pinocytic vesicles and lysosomes during heterophagy of 125I-asialofetuin by the perfused rat liver.J. Biol. Chem. 1980; 255: 5971-5978Abstract Full Text PDF PubMed Google Scholar). As shown in Fig. 2A and B, the internalization of both DiI-HDLp and OG-HDLp was prevented. Transfer of tissue that was preincubated with OG-HDLp at 4°C to medium without HDLp resulted in the formation of OG-HDLp-containing endocytic vesicles when the temperature was raised to 32°C (Fig. 2C). At 32°C, a 100-fold excess of unlabeled HDLp prevented endocytic uptake of DiI- and OG-labeled HDLp (Fig. 2D and E, respectively). These observations imply that endocytosis of HDLp by fat body cells of young adult locusts is mediated by a receptor. iLR has recently been shown to mediate endocytic uptake of HDLp in CHO cells that were stably transfected with an expression vector harboring iLR cDNA (14Van Hoof D. Rodenburg K.W. Van der Horst D.J. Insect lipoprotein follows a transferrin-like recycling pathway that is mediated by the insect LDL receptor homologue.J. Cell Sci. 2002; 115: 4001-4012Crossref PubMed Scopus (42) Google Scholar). Consequently, iLR was supposed to mediate endocytosis of HDLp in these young adult locusts. The presence of iLR was analyzed using cell membrane extracts from adult locusts at defined time points after ecdysis. Membrane proteins were separated by SDS-PAGE under reducing conditions, and immuno-detected with anti-iLR 9218 antibody raised against the cytoplasmic tail of iLR that is unique for iLRs (14Van Hoof D. Rodenburg K.W. Van der Horst D.J. Insect lipoprotein follows a transferrin-like recycling pathway that is mediated by the insect LDL receptor homologue.J. Cell Sci. 2002; 115: 4001-4012Crossref PubMed Scopus (42) Google Scholar). Under reducing conditions, iLR has a molecular weight of ∼140 kDa and is expressed in both males and females (Fig. 3A and B, respectively). In addition, the blots show that iLR is expressed during the first 3 days after imaginal ecdysis, which is in agreement with the capability of young adult fat body tissue to endocytose HDLp (Fig. 1A, B). On Day 4, expression of iLR drops below detectable levels (Fig. 3A, B), which coincides with the absence of fluorescently labeled HDLp-containing endocytic vesicles in the fat body tissue of these animals (Fig. 1I, J). Nonreduced iLR obtained from fat body cells (Fig. 3C, lane 3 and 4) has a higher electrophoretic mobility in SDS-PAGE compared with reduced iLR (Fig. 3D, lanes 3 and 4), indicating the presence of multiple disulfide bonds. The molecular weight of reduced iLR is higher than the theoretical 98 kDa based on the amino acid sequence (12Dantuma N.P. Potters M. De Winther M.P. Tensen C.P. Kooiman F.P. Bogerd J. Van der Horst D.J. An insect homolog of the vertebrate very low density lipoprotein receptor mediates endocytosis of lipophorins.J. Lipid Res. 1999; 40: 973-978Abstract Full Text Full Text PDF PubMed Google Scholar), suggesting that endogenous L. migratoria iLR is glycosylated, like all other LDLR family members (19Russell D.W. Schneider W.J. Yamamoto T. Luskey K.L. 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Wilson I.B. Marz L. Insect cells as hosts for the expression of recombinant glycoproteins.Glycoconj. J. 1999; 16: 109-123Crossref PubMed Scopus (283) Google Scholar). Immuno-detection with anti-iLR 2189/90 antibody raised against the 20 N-terminal amino acids of the first cysteine-rich repeat of iLR gave a similar result with nonreduced membrane extracts of CHO(iLR) and fat body cells as shown in Fig. 3C (Fig. 3E). These data additionally support that, despite a difference in molecular weight, the recognized membrane proteins from both cell types are iLR. RAP serves as a chaperone to assist the folding of LDLR family members and prevents premature binding of ligands in the endoplasmic reticulum (23Bu G. Marzolo M.P. Role of RAP in the biogenesis of lipoprotein receptors.Trends Cardiovasc. Med. 2000; 10: 148-155Crossref PubMed Scopus (55) Google Scholar). It has been shown to inhibit binding of ligands to lipoprotein receptors (24Herz J. Goldstein J.L. Strickland D.K. Ho Y.K. Brown M.S. 39-kDa protein modulates binding of ligands to low density lipoprotein receptor-related protein/α2-macroglobulin receptor.J. Biol. Chem. 1991; 266: 21232-21238Abstract Full Text PDF PubMed Google Scholar, 25Kounnas M.Z. Church F.C. Argraves W.S. Strickland D.K. The 39-kDa receptor-associated protein interacts with two members of the low density lipoprotein receptor family, α2-macroglobulin receptor and glycoprotein 330.J. Biol. Chem. 1992; 267: 21162-21166Abstract Full Text PDF PubMed Google Scholar, 26Battey F. Gafvels M.E. Fitzgerald D.J. Argraves W.S. Chappell D.A. Straus III, J.F. Strickland D.K. The 39-kDa receptor-associated protein regulates ligand binding by the very low density lipoprotein receptor.J. Biol. Chem. 1994; 269: 23268-23273Abstract Full Text PDF PubMed Google Scholar), including iLR (14Van Hoof D. Rodenburg K.W. Van der Horst D.J. Insect lipoprotein follows a transferrin-like recycling pathway that is mediated by the insect LDL receptor homologue.J. Cell Sci. 2002; 115: 4001-4012Crossref PubMed Scopus (42) Google Scholar). Fat body tissue of young adult locusts that was incubated with DiI-HDLp or OG-HDLp remained devoid of HDLp-containing vesicles when a 100-fold molar excess ratio of human RAP was added to the incubation medium (Fig. 4A and B, respectively). Inhibition of HDLp endocytosis by RAP suggests that the protein serves as a ligand for iLR, and thus can also be internalized by fat body cells. Incubation of young adult fat body tissue with OG-RAP resulted in a particulate pattern identical to that of endocytosed OG-HDLp (compare Figs. 4C and 1B). Endocytosis of OG-RAP was completely inhibited, with a 100-fold molar excess ratio of either unlabeled HDLp (Fig. 4D) or unlabeled RAP (Fig. 4E), suggesting that HDLp and RAP bind to the same fat body receptor. To confirm that the RAP binding receptor is iLR, fat body cell membrane proteins separated under nonreducing conditions were transferred to PVDF membrane and incubated with 125I-RAP. Immediately after adult ecdysis, fat body membrane extracts contain a single binding protein with a molecular weight of ∼110 kDa (Fig. 4F, lanes 1 and 2), which is identical to that of iLR under nonreducing conditions. These results strongly suggest that iLR is the only endocytic lipoprotein receptor expressed in this stage. In Day 4 locust fat body tissue, the RAP binding protein is no longer significantly present (Fig. 4F, lanes 3 and 4). In agreement with this finding, OG-RAP was not endocytosed by fat body tissue derived from these adult locusts (Fig. 4G). Taken together, these findings confirm that, in addition to HDLp, fat body cells are able to internalize RAP via iLR-mediated endocytosis. The finding that iLR is expressed after the imaginal ecdysis raises the question of whether similar up- and down-regulation of the receptor occurs in earlier developmental stages. Similar to adults (Fig. 5A, lanes 1 and 2), iLR is highly expressed in L5 larvae immediately after ecdysis (Fig. 5A, lanes 3 and 4), and is down-regulated on Day 4 (Fig. 5A, lanes 5 and 6). Expression of iLR in L5 larvae implies that the fat body tissue is capable of internalizing HDLp. On Day 1 after ecdysis to L5, larval fat body tissue was incubated with OG-HDLp, resulting in a particulate staining pattern (Fig. 5B) similar to that observed for young adults (Fig. 1B). On Day 4, L5 larvae have lost the ability to internalize OG-HDLp under similar conditions (Fig. 5C) [like Day 4 adults (Fig. 1J)], which coincides with the expression pattern of iLR (Fig. 5A). Collectively, these data on adults and larvae suggest that HDLp is internalized by fat body cells during the first few days after each ecdysis,
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